Functionalization of substrate surfaces with silane mixtures

ABSTRACT

Low surface energy functionalized surfaces on solid supports are provided by treating a solid support having hydrophilic moieties on its surface with a derivatizing composition containing a mixture of silanes. A first silane provides the desired reduction in surface energy, while the second silane enables functionalization with molecular moieties of interest, such as small molecules, initial monomers to be used in the solid phase synthesis of oligomers, or intact oligomers. Molecular moieties of interest may be attached through cleavable sites. Derivatizing compositions for carrying out the surface functionalization process are provided as well.

TECHNICAL FIELD

This invention relates generally to chemical functionalization ofsurfaces to modify the properties thereof. More particularly, theinvention relates to functionalization of a substrate with a silanemixture to reduce surface energy and thus constrain droplets of liquidthat are applied to the substrate surface. A primary use of theinvention is in the field of solid phase chemical synthesis,particularly solid phase synthesis of oligomer arrays.

BACKGROUND

Chemically modified, “functionalized,” solid surfaces are necessary inmany laboratory procedures involved in chemistry and biotechnology. Oneimportant application is in solid phase chemical synthesis, whereininitial derivatization of a substrate surface enables synthesis ofpolymers such as oligonucleotides and peptides on the substrate itself.Support-bound oligomer arrays, particularly oligonucleotide arrays, maybe used in screening studies for determination of binding affinity andin diagnostic applications, i.e., to detect the presence of a nucleicacid containing a specific, known oligonucleotide sequence. Modificationof surfaces for use in chemical synthesis has been described, forexample, in U.S. Pat. No. 5,624,711 to Sundberg et al., in U.S. Pat. No.5,266,222 to Willis et al., in U.S. Pat. No. 5,137,765 to Farnsworth,and in numerous other patents and publications.

In modifying siliceous or metal oxide surfaces, one technique that hasbeen used is derivatization with bifunctional silanes, i.e., silaneshaving a first functional group enabling covalent binding to the surface(often an Si-halogen or Si-alkoxy group, as in —SiCl₃ or —Si(OCH₃)₃,respectively) and a second functional group that can impart the desiredchemical and/or physical modifications to the surface. A problem withthis type of surface modification, however, is that incorporation of adesirable surface chemical functionality—provided by the secondfunctional group—may result in a surface with undesirable physicalproperties. For example, there is currently a great deal of interest insynthesizing arrays of different oligonucleotides on siliceous surfaces,and a high density of array features is generally considered desirable.The various array features can be independently created by the planarseparation of individual phosphoramidite coupling reactions as theoligonucleotides are synthesized; a simple way to achieve thisseparation is by spotting the phosphoramidite solutions onto thesurface. Feature density is then determined by the spread of thesolution droplet, which is in turn uniquely determined by both thevolume of the droplet and the contact angle between the droplet and thesurface. However, to covalently couple the first nucleotidephosphoramidite to the substrate surface requires hydroxyl moieties onthe surface, which makes the surface wettable by the phosphoramiditesolutions and thus creates droplet spread; for a given droplet volume,then, relatively large array features are provided, limiting featuredensity.

The aforementioned problem can be overcome using a variety of techniquesto constrain the droplets as they are applied to the substrate surface.Permanent wells can be formed by micromachining the substrate, with theactive surfaces subsequently modified, constraining the droplet bycapillary action. Temporary wells can also be formed using either apre-formed “stencil” or by applying a coating to the substrate andpatterning the coating. These wells could constrain the droplet byeither capillary action and/or by using a relatively unwettable coating.Alternatively, as described in U.S. Pat. No. 5,474,796 to Brennan, apattern of two different surface-bound silanes can be formed byphysically masking the surface, depositing the first silane, and thenremoving the mask and depositing the second silane. This procedure canbe used to constrain a droplet by surrounding a reactive spot on thesurface, formed by one of the two silanes, with a lower surface energyspot, formed by the other of the two silanes.

All of these procedures, however, require considerable processing andthus add substantially to the time and cost required to fabricate anarray. Also, the existence of a pattern on the substrate requires thatthe array writing apparatus be aligned with the surface pattern, anon-trivial issue for small array features.

The present invention is directed to the aforementioned need in the art,and provides a way of functionalizing substrate surfaces to reducesurface energy and thus constrain droplets of liquid that are applied tothe substrate surface, while avoiding the aforementioned problems anddifficulties associated with the procedures of the prior art.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to address theaforementioned need in the art and provide a relatively simple,straightforward process for preparing a low surface energyfunctionalized surface on a substrate.

It is an additional object of the invention to provide such a process bycoupling a mixture of silanes to hydrophilic moieties present on asubstrate surface.

It is another object of the invention to provide a process for preparingsupport-bound cleavable ligands on a low surface energy substrate,wherein the ligands may be small molecules, oligonucleotides,oligopeptides, or the like.

It is another object of the invention to provide a derivatizingcomposition for preparing a low surface energy functionalized surface ona substrate.

It is still another object of the invention to provide such aderivatizing composition comprising a mixture of silanes.

It is yet another object of the invention to provide such a derivatizingcomposition comprising a first silane that upon binding to a substratereduces the surface energy thereof, and a second silane that uponbinding to a substrate provides a means for covalently binding molecularmoieties to the substrate surface.

It is a further object of the invention to provide substrates having lowsurface energy functionalized surfaces.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

In one embodiment of the invention, then, a process is provided forpreparing a low surface energy functionalized surface on a substrate,which comprises contacting a substrate having reactive hydrophilicmoieties on its surface with a derivatizing composition comprising afirst silane R¹—Si(R^(L)R^(x)R^(y)) and a second silaneR²—(L)_(n)—Si(R^(L)R^(x)R^(y)) under reaction conditions effective tocouple the silanes to the substrate surface and provide —Si—R¹ groupsand —Si—(L)_(n)—R² groups thereon. The R^(L), which may be the same ordifferent, are leaving groups, the R^(x) and R^(y), which may also bethe same or different, are either leaving groups, like R^(L), or arelower alkyl, R¹ is a chemically inert moiety that upon binding to thesubstrate surface lowers the surface energy thereof, n is 0 or 1, L is alinking group, and R² comprises either a functional group enablingcovalent binding of a molecular moiety or a group that may be modifiedto provide such a functional group. The ratio of the silanes in thederivatizing composition determines the surface energy of thefunctionalized substrate and the density of molecular moieties that canultimately be bound to the substrate surface.

In another embodiment, a process is provided for preparing support-boundcleavable ligands on a low surface energy substrate. The processinvolves contacting a substrate having reactive hydrophilic moieties onthe surface thereof with a derivatizing composition comprising a firstsilane R¹—Si(R^(L)R^(x)R^(y)) and a second silaneR²—(L)_(n)—Si(R^(L)R^(x)R^(y)) as described above, under reactionconditions effective to couple the silanes to the substrate surface andprovide —Si—R¹ groups and —Si—(L)_(n)—R² groups thereon. A ligand isthen coupled to the surface at R², through a linking moiety containing acleavable site. The ligand may be, for example, a small molecule, afirst monomer in the solid phase synthesis of an oligomer, an intactoligomer, or the like.

In an additional embodiment, a derivatizing composition is provided forcarrying out the aforementioned processes. The derivatizing compositioncomprises a mixture of silanes, including a first silane R¹—Si(R^(L)R^(x)R^(y)) and a second silane R²—(L)_(n)—Si(R^(L)R^(x)R^(y)),wherein R¹, R², R^(L), R^(x), R^(y)and n are as defined above.

Finally, the functionalized substrates provided using the presentlydisclosed and claimed processes and compositions represent a furtherembodiment of the invention. The substrates have surface-bound —Si—R¹groups and —Si—(L)_(n)—R² groups, wherein the R¹ moieties reduce surfaceenergy and the R² moieties comprise either functional groups enablingcovalent attachment of a molecular moiety of interest or modifiablegroups that can be converted to such functional groups.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates the functionalization of a substratesurface with a derivatizing composition comprising 97.5 wt. %n-decyltrichlorosilane (“NDS”) and 2.5 wt. % undecenyltrichlorosilane(“UTS”), as described in Example 1.

FIG. 2 is a graph showing the dependence of surface hydroxyl content onthe mole ratio of UTS in the UTS/NTS derivatizing composition, evaluatedas described in Example 2.

FIG. 3 illustrates, in graph form, the increase in contact angle forvarious derivatizing compositions, including a derivatizing compositionof the invention, as described in Example 2.

FIG. 4 is a graph showing the general relationship between spot diameterand contact angle, again, as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Overview and Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,reagents, process steps, or equipment, as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, reference to “afirst silane having the structural formula R¹—Si(R^(L)R^(x)R^(y))”includes mixtures of silanes having the recited structure, while,similarly, a second silane having the structural formulaR²—(L)_(n)—Si(R^(L)R^(x)R^(y))” includes mixtures of such silanes, “acleavable site” includes a multiplicity of cleavable sites, and thelike.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “functionalization” as used herein relates to modification of asolid substrate to provide a plurality of functional groups on thesubstrate surface. By a “functionalized surface” as used herein is meanta substrate surface that has been modified so that a plurality offunctional groups are present thereon.

The terms “reactive hydrophilic site” or “reactive hydrophilic group”refer to hydrophilic moieties that can be used as the starting point ina synthetic organic process. This is contrast to “inert” hydrophilicgroups that could also be present on a substrate surface, e.g,hydrophilic sites associated with polyethylene glycol, a polyamide orthe like.

The “surface energy” γ (measured in ergs/cm²) of a liquid or solidsubstance pertains to the free energy of a molecule on the surface ofthe substance, which is necessarily higher than the free energy of amolecule contained in the in the interior of the substance; surfacemolecules have an energy roughly 25% above that of interior molecules.The term “surface tension” refers to the tensile force tending to drawsurface molecules together, and although measured in different units (asthe rate of increase of surface energy with area, in dynes/cm), isnumerically equivalent to the corresponding surface energy. By modifyinga substrate surface to “reduce” surface energy is meant lowering thesurface energy below that of the unmodified surface.

The term “monomer” as used herein refers to a chemical entity that canbe covalently linked to one or more other such entities to form anoligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptoids, and the like. In general, the monomers used inconjunction with the present invention have first and second sites(e.g., C-termini and N-termini, or 5′ and 3′ sites) suitable for bindingto other like monomers by means of standard chemical reactions (e.g.,condensation, nucleophilic displacement of a leaving group, or thelike), and a diverse element which distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid sidechain, a nucleotide base, etc.). The initial substrate-bound monomer isgenerally used as a building-block in a multi-step synthesis procedureto form a complete ligand, such as in the synthesis of oligonucleotides,oligopeptides, and the like.

The term “oligomer” is used herein to indicate a chemical entity thatcontains a plurality of monomers. As used herein, the terms “oligomer”and “polymer” are used interchangeably, as it is generally, although notnecessarily, smaller “polymers” that are prepared using thefunctionalized substrates of the invention, particularly in conjunctionwith combinatorial chemistry techniques. Examples of oligomers andpolymers include polydeoxyribonucleotides, polyribonu-cleotides, otherpolynucleotides which are—or C-glycosides of a purine or pyrimidinebase, polypeptides, polysaccharides, and other chemical entities thatcontain repeating units of like chemical structure. In the practice ofthe instant invention, oligomers will generally comprise about 2-50monomers, preferably about 2-20, more preferably about 3-10 monomers.

The term “ligand” as used herein refers to a moiety that is capable ofcovalently or otherwise chemically binding a compound of interest.Typically, when the present substrates are used in solid phasesynthesis, they are used so that “ligands” are synthesized thereon.These solid-supported ligands can then be used in screening orseparation processes, or the like, to bind a component of interest in asample. The term “ligand” in the context of the invention may or may notbe an “oligomer” as defined above. However, the term “ligand” as usedherein may also refer to a compound that is not synthesized on the novelfunctionalized substrate, but that is “pre-synthesized” or obtainedcommercially, and then attached to the substrate.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties which contain not only the known purine and pyrimidine bases,but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, or other heterocycles. In addition, the terms“nucleoside” and “nucleotide” include those moieties that contain notonly conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like.

As used herein, the term “amino acid” is intended to include not onlythe L-, D- and nonchiral forms of naturally occurring amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine), but also modified amino acids, amino acid analogs,and other chemical compounds which can be incorporated in conventionaloligopeptide synthesis, e.g., 4-nitrophenylalanine, isoglutamic acid,isoglutamine, ε-nicotinoyl-lysine, isonipecotic acid,tetrahydroisoquinoleic acid, α-aminoisobutyric acid, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, 4-aminobutyric acid, and the like.

The terms “protection” and “deprotection” as used herein relate,respectively, to the addition and removal of chemical protecting groupsusing conventional materials and techniques within the skill of the artand/or described in the pertinent literature; for example, reference maybe had to Greene et al., Protective Groups in Organic Synthesis, 2ndEd., New York: John Wiley & Sons, 1991. Protecting groups prevent thesite to which they are attached from participating in the chemicalreaction to be carried out.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well ascycloalkyl groups such as cyclopentyl, cyclohexyl and the like. The term“lower alkyl” intends an alkyl group of 1 to 6 carbon atoms, preferably1 to 4 carbon atoms.

The term “alkoxy” as used herein refers to a substituent —O—R wherein Ris alkyl as defined above. The term “lower alkoxy” refers to such agroup wherein R is lower alkyl.

The term “alkylene” as used herein refers to a difunctional saturatedbranched or unbranched hydrocarbon chain containing from 1 to 24 carbonatoms, and includes, for example, methylene (—CH₂—), ethylene(—CH₂—CH₂—), propylene (—CH₂—CH₂—CH₂—), 2-methylpropylene(—CH₂—CH(CH₃)—CH₂—), hexylene (—(CH₂)₆—), and the like. “Lower alkylene”refers to an alkylene group of 1 to 6, more preferably 1 to 4, carbonatoms.

The terms “alkenyl” and “olefinic” as used herein refer to a branched orunbranched hydrocarbon group of 2 to 24 carbon atoms containing at leastone carbon-carbon double bond, such as ethenyl, n-propenyl, isopropenyl,n-butenyl, isobutenyl, t-butenyl, octenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like.

The terms “halogen” or “halo” are used in the conventional sense torefer to a chloro, bromo, fluoro or iodo substituent. “Optional” or“optionally” means that the subsequently described circumstance may ormay not occur, so that the description includes instances where thecircumstance occurs and instances where it does not. For example, thephrase “optionally substituted” means that a non-hydrogen substituentmay or may not be present, and, thus, the description includesstructures wherein a non-hydrogen substituent is present and structureswherein a non-hydrogen substituent is not present.

Accordingly, the invention in a first embodiment is directed to aprocess for preparing a low surface energy functionalized surface on asubstrate. The functionalized surface prepared using this process hasfunctional groups enabling covalent binding of molecular moieties, suchas in solid phase chemical synthesis or the like, but nevertheless haslowered surface energy so that wettability is reduced and liquiddroplets applied to the substrate surface are constrained (i.e., do notspread to the extent that they would in the absence of the presentlydisclosed and claimed surface modification process).

The inventive process involves contacting the surface of a solidsubstrate with a derivatizing composition that contains a mixture ofsilanes, under reaction conditions effective to couple the silanes tothe substrate surface via reactive hydrophilic moieties present on thesubstrate surface. The reactive hydrophilic moieties on the substratesurface are typically hydroxyl groups, carboxyl groups, thiol groups,and/or substituted or unsubstituted amino groups, although, preferably,the reactive hydrophilic moieties are hydroxyl groups. The substrate maycomprise any material that has a plurality of reactive hydrophilic siteson its surface, or that can be treated or coated so as to have aplurality of such sites on its surface. Suitable materials include, butare not limited to, supports that are typically used for solid phasechemical synthesis, e.g., cross-linked polymeric materials (e.g.,divinylbenzene styrene-based polymers), agarose (e.g., Sepharose®),dextran (e.g., Sephadex®), cellulosic polymers, polyacrylamides, silica,glass (particularly controlled pore glass, or “CPG”), ceramics, and thelike. The supports may be obtained commercially and used as is, or theymay be treated or coated prior to functionalization.

The derivatizing composition contains two types of silanes, a firstsilane that may be represented as R¹—Si(R^(L)R^(x)R^(y)) and a secondsilane having the formula R²—(L)_(n)—Si(R^(L)R^(x)R^(y)). In theseformulae, the R^(L), which may be the same or different, are leavinggroups, the R^(x)and R^(y), which may be the same or different, areeither lower alkyl or leaving groups like R^(L), R¹ is a chemicallyinert moiety that upon binding to the substrate surface lowers thesurface energy thereof, n is 0 or 1, L is a linking group, and R² is afunctional group enabling covalent binding of a molecular moiety or agroup that may be modified to provide such a functional group. Reactionof the substrate surface with the derivatizing composition is carriedout under reaction conditions effective to couple the silanes to thesurface hydrophilic moieties and thereby provide —Si—R¹ groups and—Si—(L)_(n)—R² groups on the substrate surface.

More specifically, the R^(L) moieties, which are leaving groups, aresuch that they enable binding of the silanes to the surface. Typically,the leaving groups are hydrolyzable so as to form a silanol linkage tosurface hydroxyl groups. Examples of suitable leaving groups include,but are not limited to, halogen atoms, particularly chloro, and alkoxymoieties, particularly lower alkoxy moieties. The R^(x) and R^(y) areeither lower alkyl, e.g., methyl, ethyl, isopropyl, n-propyl, t-butyl,or the like, or leaving groups as just described with respect to R^(L).Thus, each type of silane will generally contain a trichlorosilylfunctionality, a tri(lower)alkoxysilyl functionality such astrimethoxysilyl, mixed functionalities such as diisopropylchlorosilyl,dimethylchlorosilyl, ethyldichlorosilyl, methylethylchlorosilyl or thelike.

The first silane is the derivatizing agent that reduces surface energyas desired, while the second silane provides the surfacefunctionalization necessary for covalent attachment of an additionalmolecular moiety, e.g., a ligand, a monomer, an oligomer, etc. Thus,with respect to the first silane, coupling to the substrate yieldssurface —Si—R¹ groups as explained above, wherein R¹ is a chemicallyinert moiety that upon binding to the substrate surface lowers surfaceenergy. By “chemically inert” is meant that R¹ will not be cleaved ormodified when the functionalized substrate is used for its intendedpurpose, e.g., in solid phase chemical synthesis, hybridization assays,or the like. Typically, R¹ is an alkyl group, generally although notnecessarily containing in the range of 2 to 24 carbon atoms, preferablyin the range of 10 to 18 carbon atoms. R¹ may also be benzyl, eitherunsubstituted or substituted with 1 to 5, typically 1 to 3, halogen,preferably fluoro, atoms.

The second silane, upon coupling, provides surface —Si—(L)_(n)—R²groups. Of course, if the R^(x) and R^(y) are not leaving groups, thesurface moieties provided will actually be “—SiR^(x)R^(y)—(L)_(n)—R²”groups, which applicants intend to encompass by the more genericrepresentation “—Si—(L)_(n)—R² ”. R² comprises either a functional groupthat can bind directly to an additional molecular species of interest,or a modifiable group that can be converted to such a functional groupunder conditions that do not substantially affect any other chemicalspecies that are present. That is, R² may be a functional group such ashydroxyl, carboxyl, amino, or the like, or it may be a modifiable groupsuch an olefinic moiety, e.g., a terminal —CH═CH₂ group, which canreadily be converted to a reactive hydroxyl group by boration andoxidation using procedures known in the art. L represents a linker and nis 0 or 1, such that a linker may or may not be present. If a linker ispresent, it will generally be a C₁-C₂₄ hydrocarbylene linking group.Normally, L is C₁-C₂₄ alkylene, preferably C₁₀-C₁₈ alkylene.

The density of R² groups on the substrate surface, following reactionwith the derivatizing composition, is determined by the relativeproportions of the first and second silanes in the derivatizingcomposition. That is, a higher proportion of the second silane in thederivatizing composition will provide a greater density of R² groups,while a higher proportion of the first silane will give rise to a lowerdensity of R² groups. Optimally, the first silane represents in therange of approximately 0.5 wt. % to 50 wt. % of the derivatizationcomposition, preferably in the range of approximately 1.0 wt. % to 10wt. % of the composition, while the second silane correspondinglyrepresents in the range of approximately 50 wt. % to 99.5 wt. % of thederivatization composition, preferably in the range of approximately 90wt. % to 99 wt. % of the composition.

Functionalized substrates prepared using the aforementioned proceduresare believed to be novel and are claimed as such herein. The surface ofthe functionalized substrates contain both —Si—R¹ and Si—(L)_(n)—R²groups, present at a predetermined ratio, with the ratio determiningboth surface energy and density of functional groups. These substratesmay be used, for example, in any of a number of known chemical andbiological procedures, such as in solid phase chemical synthesis, e.g.,of oligonucleotides, oligopeptides, and oligosaccharides, in thepreparation of combinatorial libraries, in chemical separationprocedures, in screening processes, and the like. Such procedures are incurrent use and will thus be known to those skilled in the art and/ordescribed in the pertinent literature and texts. For example, synthesisof polynucleotide libraries using now conventional phosphoramidite orphosphotriester chemistry is described by Beaucage et al. (1981)Tetrahedron Lett. 22:1859-62, and Itakura et al. (1975) J. Biol. Chem.250:4592 (1975). Houghten (1985) Proc. Natl. Acad. Sci. USA82:5131-5135), describes the preparation of a combinatorial library ofpeptides using a modification of the Merrifield method (Merrifield(1963) J. Am. Chem. Soc. 85:2149-2154; Tam et al., The Peptides (NewYork: Academic Press, 1975), at pp. 185-249); and OligonucleotideSynthesis, M. J. Gait, Ed. (New York: IRL Press, 1990).

For example, synthesis of support-bound oligonucleotides is normallyconducted by successive addition of protected nucleotides to a growingoligonucleotide chain, wherein the terminal 5′ hydroxyl group is causedto react with adeoxyribonucleoside-3′—O—(N,N-diisopropylamino)phosphoramidite protectedat the 5′ position with dimethoxytrityl or the like, the 5′ protectinggroup is removed after the coupling reaction, and the procedure isrepeated with additional protected nucleotides until synthesis of thedesired oligonucleotide is complete.

Additionally, and as will be appreciated by those skilled in the art,oligopeptide synthesis on a support—as may be carried out herein byvirtue of the support-bound R² substituent—involves sequential additionof carboxyl-protected amino acids to a growing peptide chain, with eachadditional amino acid in the sequence similarly protected and coupled tothe terminal amino acid of the oligopeptide under conditions suitablefor forming an amide linkage. After oligopeptide synthesis is complete,acid is used to remove the remaining terminal protecting groups. Thesupport-bound oligopeptides thus provided can then be used in any numberof ways, e.g., in screening procedures involved in combinatorialprocesses, in chromatographic methods, and the like.

In an alternative embodiment, the method and reagents of the inventionare used to provide oligomers bound to the support via a chemicallycleavable site. That is, in this alternative process, following reactionof the substrate surface with the first and second silanes, a furtherreaction is conducted at R². This reaction involves reaction of R² witha linking group containing a cleavable site, such as an ester group, andthe free terminus of the bound linking group is then used for solidphase synthesis. Conversion of R² to a different moiety may or may notbe desired prior to attaching the linking group. For example, R² may bean alkylamino substituent, in which case the amino moiety serves as thereactive site for binding the linking group, or R² may be bromo, inwhich case it is desirable to convert R² to a primary or secondary aminosubstituent, and then carry out the reaction to the linking group. Inthis way, the bound ligand, monomer, oligomer, or the like may becleaved from the solid support by treatment of the surface with anappropriate reagent.

Suitable cleavable sites include, but are not limited to, the following:base-cleavable sites such as esters, particularly succinates (cleavableby, for example, ammonia or trimethylamine), quaternary ammonium salts(cleavable by, for example, diisopropylamine) and urethanes (cleavableby aqueous sodium hydroxide); acid-cleavable sites such benzyl alcoholderivatives (cleavable using trifluoroacetic acid), teicoplanin aglycone(cleavable by trifluoroacetic acid followed by base), acetals andthioacetals (also cleavable by trifluoroacetic acid), thioethers(cleavable, for example, by HF or cresol) and sulfonyls (cleavable bytrifluoromethane sulfonic acid, trifluoroacetic acid, thioanisole, orthe like); nucleophile-cleavable sites such as phthalamide (cleavablewith substituted hydrazines), esters (cleavable with, for example,aluminum trichloride) and Weinreb amide (cleavable with lithium aluminumhydride); and other types of chemically cleavable sites, includingphosphorothioate (cleavable with silver or mercuric ions) anddiisopropyldialkoxysilyl (cleavable with fluoride ion). Other cleavablesites will be apparent to those skilled in the art or are described inthge pertinent literature and texts (e.g., Brown (1997) ContemporaryOrganic Synthesis 4(3):216-237).

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description as well as the example that follows are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All patents and publications mentioned herein, both supra and infra, arehereby incorporated by reference.

EXAMPLE 1 PREPARATION OF FUNCTIONALIZED SURFACES

This example describes functionalization of a glass substrate with aderivatizing composition comprising 97.5 wt. % n-decyltrichlorosilane(“NTS”) as a first silane and 2.5 wt. % undecenyltrichlorosilane (“UTS”)as a second silane, followed by boration and oxidation to convert theterminal olefinic moiety of the surface-bound UTS to a hydroxyl group.This procedure is shown schematically in FIG. 1. Evaluation of thefunctionalized surface is also described.

(a) Silylation:

Under moisture-free conditions, 14 ml NTS and 0.4 ml UTS were added to800 ml of anhydrous toluene, and swirled to mix. Cleaned glasssubstrates were placed into a ca. 1 liter reactor equipped for inert gaspurging, heating and stirring, and purging was conducted for 30 minutes.Moisture-free conditions were maintained, and the NTS/UTS solution wasadded to the reactor. The solution was heated to 100° C. for 4 hours,while stirring and continuing to maintain moisture-free conditions. Thesilane solution was removed from the reactor and replaced with anhydroustoluene. This step was repeated twice.

The substrates were then removed from the reactor and rinsed rigorouslywith an appropriate solvent. The bulk solvent was removed from thesubstrates by blowing with clean inert gas. The substrates were placedin a vacuum oven preheated to 150° C. and heated under vacuum for 1hour. The substrates were removed and allowed to cool to ambienttemperature.

(b) Boration and Oxidation:

The silylated substrates prepared in part (a) were placed in a ca. 1liter reactor equipped for inert gas purging and stirring, and purgingwas conducted for 30 minutes. Under moisture-free conditions, 800 ml of1.0 M borane-tetrahydrofuran complex was transferred to the reactor. Thesubstrates were incubated while stirring, for two hours. Then, whilemaintaining moisture-free conditions, the boration solution was removedand replaced with 800 ml anhydrous tetrahydrofuran. The substrates wereremoved and rinsed rigorously with an appropriate solvent. Bulk solventwas removed by blowing with clean inert gas.

To a 1 liter vessel equipped for stirring, 800 ml of 0.1 N NaOH in 30%hydrogen peroxide (aqueous) was added. The oxidized substrates wereimmersed therein, and incubated, with stirring, for 10 minutes. Thesubstrates were removed and rinsed rigorously with an appropriatesolvent, then dried by blowing with clean inert gas.

The processes of steps (a) and (b) were repeated using different moleratios of NTS and UTS, 100% UTS, and a mixture of glycidoxypropyltrimethoxysilane and hexaethylene glycol (GOPS-HEG). This hydroxylsilane-linker was prepared following the procedure of Maskos et al.(Maskos et al. (1992) Nucleic Acids Res. 20:1679) who demonstrated it tobe useful for both oligonucleotide synthesis and hybridization.

EXAMPLE 2 EVALUATION OF FUNCTIONALIZED SURFACES

Surface hydroxyl density (molecules/μm²) of the functionalized surfacesprepared in Example 1 was evaluated spectrophotometrically, and FIG. 2shows the dependence of surface hydroxyl content on the mole ratio ofUTS in the UTS/NTS derivatizing composition. FIG. 3 shows the increasein contact angle for several UTS mole ratios and two solvents ofinterest, acetonitrile and adiponitrile, in comparison to a GOPS-ethanolmixture. Contact angles reported are static contact angle measurementsas described in the literature (Chan, Chi-Ming, Polymer SurfaceModification and Characterization, chapter 2 (New York: HansaPublishers, 1993). Measurements were performed on 25 μl aliquots of theappropriate solvent using an FTA200 instrument (First Ten Angstroms,South San Francisco, Calif.). FIG. 4 shows the general relationshipbetween the spot diameter and contact angle. For 100 nl drops, thefollowing spot diameters were observed for the GOPS-ethanol mixture andthe 2.5% UTS/NTS derivatizing composition:

GOPS-ethanol 2.5% UTS/NTS acetonitrile  >5 mm 1.4 mm adiponitrile 2.7 mm0.9 mm

Thus, the derivatizing composition of the invention significantlyreduces spot diameter for a droplet of a given volume.

What is claimed is:
 1. A substrate having a functionalized surface,comprising: a solid support having a plurality of surface hydrophilic,nucleophilic groups, a first fraction of which are covalently bound toan —Si—R¹ moiety and a second fraction of which are covalently bound toan —Si—(L)_(n)—R² moiety, wherein R¹ is a chemically inert moiety thatlowers the surface energy of the solid support and is selected from thegroup consisting of C₁₀ to C₁₈ alkyl and benzyl optionally substitutedwith 1 to 5 halogen atoms, n is 0 or 1, L is a linking group, R² is afunctional group enabling covalent binding of a molecular moiety or amodifiable group that can be converted to such a functional group, andthe solid support is comprised of a material selected from the groupconsisting of polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, and glass.
 2. A substrate having a functionalizedsurface, comprising: a solid support having a plurality of surfacehydrophilic, nucleophilic groups, a first fraction of which arecovalently bound to an —Si—R¹ moiety and a second fraction of which arecovalently bound to an —Si(R^(x)R^(y))—L—R² moiety, wherein R^(x) andR^(y) are independently selected from the group consisting of halogen,lower alkoxy and lower alkyl, R¹ is C₁₀ to C18 alkyl, L is C₁-C₂₄alkylene, and R² is —CH═CH₂.
 3. A process for functionalizing thesurface of a substrate, comprising: contacting a substrate havingreactive hydrophilc moieties on the surface there with a derivatizingcomposition comprising a first silane R¹—Si(R^(L)R^(x)R^(y)) and asecond silane R²—(L)_(n)—Si(R^(L)R^(x)R^(y)) under reaction conditionseffective to couple the silanes to the substrate surface and provide—Si—R¹ groups and —Si—(L)_(n)—R² groups thereon, wherein the R^(L),moieties, which may be the same or different, are leaving groups, theR^(x) and R^(y) are independently lower alkyl or leaving groups, R¹ is achemically inert moiety that upon binding to the substrate surfacelowers the surface energy thereof and is selected from the groupconsisting of C₁₀ to C₁₈ alkyl and benzyl optionally substituted with 1to 5 halogen atoms, n is 0 or 1, L is a linking group, and R² is afunctional group enabling covalent binding of a molecular moiety or amodifiable group that may be converted to such a functional group. 4.The process of claim 1, wherein the reactive hydrophilic moieties areselected from the group consisting of hydroxyl, carboxyl, thiol, amino,and combinations thereof.
 5. The process of claim 2, wherein thereactive hydrophilic moieties are hydroxyl groups.
 6. The process ofclaim 1, wherein the R^(L) are selected from the group consisting ofhalogen and alkoxy.
 7. The process of claim 6, wherein the R^(L) areselected from the group consisting of chloro and lower alkoxy.
 8. Theprocess of claim 3, wherein R¹ is C₁₀ to C₁₈ alkyl. 9.The process ofclaim 1, wherein n is
 1. 10. the process of claim 9, wherein L is aC₁-C₂₄ hydrocarbylene linking group substituted with 0 to 6 substituentsselected from the group consisting of lower alkyl, hydroxyl, halogen andamino, optionally containing 1 to 6—O—, —S—, —NR—, —CONH—, —(CO)— or—COO— linkages wherein R is hydrogen or lower alkyl.
 11. The process ofclaim 10, wherein L is C₁-C₂₄ alkylene.
 12. The process of claim 11,wherein L is C₁₀-C₁₈ alkylene.
 13. The process of claim 1, wherein R² is—CH═CH₂.
 14. The process of claim 1, wherein, in the first silane, R^(x)and R^(y) are lower alkyl.
 15. The process of claim 1, wherein, in thesecond silane, R^(x) and R^(y) are lower alkyl.
 16. A process forfunctionalizing the surface of a substrate, comprising: contacting asubstrate having reactive hydrophilic moieties on the surface thereofwith a derivatizing composition comprising a first silaneR¹—Si(R^(L)R^(x)R^(y)) and a second silane R²—L—Si(R^(L)R^(x)R^(y))under reaction conditions effective to couple the silanes to thesubstrate surface and provide —Si—L—R¹ groups and —Si—L—R² groupsthereon, wherein the R^(L) moieties are independently selected from thegroup consisting of halogen and lower alkoxy, R^(x) and R^(y) areindependently selected from the group consisting of halogen, loweralkoxy and lower alkyl, R¹ is C₁₀ to C₁₈ alkyl, L is C₁-C₂₄ alkylene,and R² is —CH═CH₂.
 17. The process of claim 16, further comprisingconverting R² to a hydroxyl group by boration and oxidation.
 18. Aprocess for functionalizing the surface of a substrate, comprising:contacting a substrate having reactive hydrophilic moieties on thesurface thereof with a derivatizing composition comprising a firstsilane R¹—Si(R^(L)R^(x)R^(y)) and a second silaneR²—(L)_(n)—Si(R^(L)R^(x)R^(y)) under reaction conditions effective tocouple the silanes to the substrate surface and provide —Si—R¹ groupsand —Si—(L)_(n)—R² groups thereon, wherein the R^(L), moieties, whichmay be the same or different, are leaving groups, the R^(x) and R^(y)are independently lower alkyl or leaving groups, R¹ is a C₁₀ to C₁₈chemically inert moiety that upon binding to the substrate surfacelowers the surface energy thereof, n is 0 or 1, L is a linking group,and R² is a functional group enabling covalent binding of a molecularmoiety or a modifiable group that may be converted to such a functionalgroup.
 19. A derivatizing composition for functionalizing the surface ofa substrate, comprising: a first silane R¹—Si(R^(L)R^(x)R^(y)) and asecond silane R²—(L)_(n)—Si(R^(L)R^(x)R^(y)), wherein the R^(L) areindependently leaving groups, the R^(x) and R^(y) may be the same ordifferent and are either lower alkyl or leaving groups, R¹ is achemically inert moiety that upon binding to a substrate surface lowersthe surface energy thereof and is selected from the group consisting ofC₁₀ to C₁₈, alkyl and benzyl optionally substituted with 1 to 5 halogenatoms, n is 0 or 1, L is a linking group, and R² is a functional groupenabling covalent binding of a molecular moiety or a modifiable groupthat can be converted to such a functional group.
 20. A derivatizingcomposition for functionalizing the surface of a substrate, comprising afirst silane R¹—Si(R^(L)R^(x)R^(y)) and a second silaneR²—L—Si(R^(L)R^(x)R^(y)), wherein the R^(L) moieties are independentlyselected from the group consisting of halogen and lower alkoxy, R^(x)and R^(y) are independently selected from the group consisting ofhalogen, lower alkoxy and lower alkyl, R¹ is C₁₀ to C₁₈ alkyl, L isC₁-C₂₄ alkylene, and R² is —CH═CH₂.